ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

Abstract

The proliferative action of ERalpha largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERbeta displays tumor-suppressor properties, thus supporting the interest to identify compounds that could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) upregulated ERbeta protein levels, whereas it decreased ERalpha expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERbeta more strongly than that of ERalpha. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERbeta expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERbeta and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy.

Fig. 1. ERα and ERβ levels are differentially regulated by HDI

A and B. MCF-7 cells were pretreated or not with cycloheximide (CHX) at 20μg/ml for 2 h and then further stimulated for 4 hrs with TSA (500 ng/ml) with CHX still present or not. Control cells were treated in parallel with ethanol alone in the presence or absence of CHX. Total RNA was extracted and the expression of ERα (panel A) and ERβ (panel B) mRNAs was quantified by real-time PCR. Results represent the mean of 2 independent quantifications. ERα protein levels were determined by western blot on cells treated or not with TSA. C. ERβ mRNA and protein levels were determined in OVCAR-3 cells treated or not with TSA.

Fig. 2. TSA differentially regulates ERα and ERβ activities

A. MDA-MB-231, HeLa, PEO-14, HEK-293 cells were transfected with ERE2-TK-LUC reporter construct along with CMV5, CMV-hERα or CMV-hERβ expression vectors. Cells were then treated with control vehicle ethanol (C), estradiol (E2 10⁻⁸M), TSA (500 ng/ml) or the combination of E2 and TSA for 18h prior to harvesting cells for luciferase assays. Results show relative luciferase activities (expressed as percent of the values obtained in the absence of E2 for each conditions) after normalization for β-galactosidase activity (n=3 independent experiments). The levels of expressed receptors have been monitored by western blots. B. Same experiment as in A but in HeLa cells transfected with pS2 promoter C. HeLa cells transfected as in A were then treated with E2 (10⁻⁸M) and either ethanol vehicle (C), sodium butyrate (NaBu, 2 mM) or SAHA (5μM) for 18h. Results show relative luciferase activities (% of values without E2) after normalization for β-galactosidase activity (n=3 independent experiments).

Fig. 3. Regulation of ER activity by HDI is a general phenomenon

A. HeLa cells were transfected with ERE2-TK-LUC reporter construct along with CMV-5 (●) CMV-hERα (■) or CMV-hERβ (△) expression vectors and treated with ethanol control vehicle (C) or increasing concentrations of E2, without or with TSA (500 ng/ml). Results are expressed as percent of values obtained in the absence of E2. B and C. Same conditions as in A to analyze the effects of E2 (10⁻⁸M), propyl pyrazole triol (PPT, 10⁻⁸M), diarylpropionitrile (DPN, 10⁻⁸M), hydroxy-tamoxifen (OHTam, 10⁻⁸M) or ICI 182,780 (ICI, 10⁻⁸M).

Fig. 4. ERα and ERβ modulate the transcriptional regulation by TSA

A. Representation of the deletion mutants used. B. Wild-type or deletion mutants of hERα and hERβ were transfected in HeLa cells along with ERE2-TK-LUC construct. Cells were then treated with TSA (500 ng/ml) in the presence (upper panel) or absence (lower panel) of estradiol (E2 10⁻⁸M) for 18h prior to harvesting cells for luciferase assays. Results show relative luciferase activities (expressed as percent of the values obtained in the absence of TSA for each conditions) after normalization for β-galactosidase activity (n=3 independent experiments). The dotted lines show the level of luciferase obtained in the absence or presence of TSA in CMV5-transfected cells.

Fig. 5. Expression of ERβ drastically enhances the anti-proliferative effects of TSA

A. HeLa cells were either infected with Ad5, Ad-hERα, or Ad-hERβ viruses at MOI 100. The cells were treated with ethanol vehicle or TSA (50 ng/ml) 24 h after the beginning of the infection. Proliferation rate was determined by counting the cells at days 4. Results are express as % of Ad5-infected cells treated with ethanol alone and represent the mean ± SD of four independent determinations. B. Same proliferation experiments performed in MDA-MB-231 cells. C. MDA-MB-231 cells which had been infected as in A, were then treated for 24 h with control vehicle Ethanol (C) or TSA (50 ng/ml). Apoptosis was set to 100% in control cells (not treated with TSA) infected with Ad5, Ad-ERα or Ad-ERβ viruses. Results represent the mean of ± SD of three independent determinations.

Fig. 6. ERα and ERβ differentially regulate genes involved in proliferation

A. HeLa cells were transfected with the following reporter constructs: p21^WAF1/CIP1-Luc, cycD1-Luc, cyclinE-Luc or (TRE)5-Luc (AP-1), along with hERα and hERβ expression vectors. Cells were then treated (hatched boxes) or not (empty boxes) with TSA (500 ng/ml) for 18h prior to harvesting cells for luciferase assays. Results show relative luciferase activities (n=3 independent experiments) after normalization for β-galactosidase activity. B–C. HeLa cells were transfected with CMV-hERα or CMV-hERβ expression vectors. Cells were treated for 20h with ethanol vehicle or TSA (500 ng/ml). Cyclin E (B) and p21^WAF1/CIP1 (C) protein expression was monitored by western blot.